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ABI3 and PIL5 Collaboratively Activate the Expression ofSOMNUS by Directly Binding to Its Promoter in ImbibedArabidopsis Seeds W
Jeongmoo Park,1 Nayoung Lee,1 Woohyun Kim, Soohwan Lim, and Giltsu Choi2
Department of Biological Sciences, KAIST, Daejeon 305-701, Korea
A previous study showed that SOMNUS (SOM), which encodes a C3H-type zinc finger protein, is a key negative regulator of
seed germination that acts downstream of PHYTOCHROME INTERACTING FACTOR3-LIKE5 (PIL5). However, it was not
determined if PIL5 is the sole regulator of SOM expression. Public microarray data suggest that the expression of SOM
mRNA is regulated also by ABSCISIC ACID INSENSITIVE3 (ABI3), another key regulator of seed germination. By analyzing
abi3 mutants and ABI3 overexpression lines, we show here that ABI3 activates the expression of SOM mRNA collaboratively
with PIL5 in imbibed seeds. Chromatin immunoprecipitation analysis coupled with electrophoretic mobility shift assay
indicate that ABI3 activates the expression of SOM mRNA by directly binding to two RY motifs present in the SOM promoter
in vivo, which is further supported by the greatly decreased expression of a reporter gene driven by a SOM promoter bearing
mutated RY motifs. At the protein level, the ABI3 protein interacts with the PIL5 protein. The ABI3-PIL5 interaction, however,
does not affect targeting of ABI3 and PIL5 to SOM promoters. Taken together, our results indicate that ABI3 and PIL5
collaboratively activate the expression of SOM mRNA by directly binding to and interacting with each other at the SOM
promoter.
INTRODUCTION
The decision for a seed to germinate at a given time and space is
determined by seed developmental status and environmental
conditions. In Arabidopsis thaliana, freshly harvested seeds
display seed dormancy, a property that inhibits germination
even in favorable environmental conditions (Finch-Savage and
Leubner-Metzger, 2006). Abscisic acid (ABA) plays important
roles in both initiating andmaintaining seed dormancy, as shown
by the shallow seed dormancy of ABA syntheticmutants, such as
aba2, and the deep seed dormancy of ABA catabolic mutants,
such as a cyp707a triple mutant (Leon-Kloosterziel et al., 1996;
Okamoto et al., 2010). Mutations in some of the ABA signaling
genes, such as ABSCISIC ACID INSENSITIVE3 (ABI3), which
encodes a DNA binding protein that acts as a positive component
of ABA signaling, also disrupts seed dormancy (Lopez-Molina
et al., 2002). Other components, such as DELAY OF GERMINA-
TION1 and HISTONEMONOUBIQUITINATION1, have been iden-
tified as being important regulators of seed dormancy, but their
relationshipwithABA signaling has not beendetermined (Bentsink
et al., 2006; Liu et al., 2007).
Freshly harvested seeds eventually lose dormancy when they
are sufficiently dried or have undergone stratification. It is not
clear how seed dormancy breaks, but, once broken, nondormant
seeds germinate if environmental conditions are favorable.
Among various environmental factors, light and temperature
play important roles in the decision to germinate. A seed must
monitor and integrate various environmental conditions into
cellular processes in order for germination to occur. Ultimately,
favorable conditions activate germination-promoting hormone
signaling, such as gibberellic acid (GA) signaling, and repress
germination-inhibiting hormone signaling, such as ABA signal-
ing. The molecular pathways involved in monitoring environmen-
tal conditions and integrating them with hormonal signaling are
currently being actively investigated (Finkelstein et al., 2008;
Holdsworth et al., 2008; Seo et al., 2009).
Phytochrome-mediated light signaling provides a good model
system of how environmental conditions are perceived and
integrated into internal cellular processes. Inside seed cells,
phytochromes perceive red and far-red spectra of light. In
Arabidopsis, phytochrome B (phyB), which is the major phytoh-
crome present in dry seeds, perceives red light and promotes
seed germination. Phytochrome A (phyA), which accumulates
during seed imbibition, also perceives light and promotes seed
germination. Unlike phyB, however, phyA perceives both very
low fluences of all spectra of visible light and prolonged far-red
light (Shinomura et al., 1996). Other minor phytochromes, such
as phyE, also play a role in promoting seed germination in
imbibed seeds (Hennig et al., 2002). When imbibed seeds
perceive light, activated phytochromes enter the nucleus and
transmit the light signal, partly by destabilizing a germination-
inhibiting, phytochrome-interacting bHLH transciption factor,
called PIL5 (also known as PIF1) (Oh et al., 2004, 2006). The
central role of PIL5 in mediating phytohcrome signaling can be
1 These authors contributed equally to this work.2 Address correspondence to gchoi@kaist.edu.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Giltsu Choi(gchoi@kaist.edu).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.110.080721
The Plant Cell, Vol. 23: 1404–1415, April 2011, www.plantcell.org ã 2011 American Society of Plant Biologists
inferred from the 100%germination frequency of the pil5mutant,
even if phytochrome B is inactivated by far-red light. In addition,
microarray analysis indicates that all genes in imbibed seeds that
are differentially expressed in response to red light are either
directly or indirectly regulated by PIL5 (Oh et al., 2009). Thus, the
destabilization of PIL5 by activated phytohcromes serves to
release the repression of seed germination imposed by PIL5 and
allows seeds to germinate.
PIL5 inhibits seed germination both by coordinating various
hormone signaling cascades and by inhibiting cell wall loosening
in imbibed seeds (Oh et al., 2009). Chromatin immunoprecipita-
tion analysis coupled with microarray analysis shows that PIL5
regulates the expression of 166 genes by directly binding to their
promoters. These direct target genes include cell wall loosening
genes, such as expansin and xyloglucan endotransglycosylase
genes, and various hormone signaling genes, such as GA sig-
naling genes (GA INSENSITIVE [GAI] and REPRESSOR OF GA
[RGA]), ABA signaling genes (ABI5 and ABI3), auxin signaling
genes (AUXIN RESPONSE FACTOR18 [ARF18] and INDOLE
ACETIC ACID-INDUCED PROTEIN16 [IAA16]), cytokinin signal-
ing genes (CYTOKININ RESPONSE FACTOR1 [CRF1], CRF2,
and CRF3), a JA signaling gene (JAZ1), and a BR signaling gene
(BES1-INTERACTING MYC-LIKE PROTEIN2). The direction of
expression indicates that PIL5 inhibits seed germination partly by
activating the expression of GAI and RGA, which inhibit seed
germination as negative GA signaling components, and of ABI3
and ABI5, which also inhibit seed germination as positive ABA
signaling components. The role of other signaling components in
seed germination is not clearly defined. However, the repression
of JAZ1 and IAA16, negative signaling components of JA and
auxin signaling, respectively, and the activation of ARF8, a
positive auxin signaling component, are also consistent with
the inhibitory role of PIL5 in seed germination. PIL5 also mod-
ulates hormone signaling by regulating themetabolism of various
hormones. For example, PIL5 decreases the level of GA by re-
pressing GA synthetic genes (GA3ox1 and GA3ox2) and activat-
ing a GA catabolic gene (GA2ox1), while it increases the ABA level
by activating ABA synthetic genes (ABA DEFICIENT1 [ABA1],
NINE-CIS-EPOXYCAROTENOID DIOXYGENASE6 [NCED6], and
NCED9) and repressing an ABA catabolic gene (CYP707A2). The
reciprocal regulation of hormone synthetic and catabolic genes
can also be seen in auxin metabolism, as PIL5 activates the
expression of genes involved in auxin synthesis (NITRILASE1
[NIT1], NIT3, ALDEHYDE OXIDASE1, and AMIDASE1) but re-
presses the expression of a gene involved in auxin catabolism
(DWARF IN LIGHT1). Curiously, PIL5 does not bind to the pro-
moters of most genes involved in hormone metabolism, suggest-
ing that PIL5 indirectly regulates these metabolic genes through
other direct target genes. However, the degree of separation
between PIL5 and genes involved in hormone metabolism is not
known. Furthermore, genetic networks operating under PIL5 to
regulate seed germination have not yet been identified. An anal-
ysis of the genes directly targeted by PIL5 might be useful to
identify these genetic networks.
As a positive ABA signaling component, ABI3 also regulates
various aspects of seed development (Giraudat et al., 1992). It
belongs to a plant-specific B3 domain protein family that in-
cludes ABI3, FUSCA3 (FUS3), and LEAFY COTYLEDON2 (LEC2)
in Arabidopsis and VIVIPAROUS1 (VP1) in maize (Zea mays).
ABI3 contains four domains, dubbed A, B1, B2, and B3, which
are named after the activation domain and the three basic amino
acid clusters contained in it (Bies-Etheve et al., 1999). Among
them, the B3 domain is known to bind to the RY motif (CATGCA)
in vitro (Suzuki et al., 1997; Monke et al., 2004; Braybrook et al.,
2006), while the B1 and B2 domains interact with a set of bZIP
transcription factors, including ABI5, bZIP10, bZIP25, and
TRAB1 (Hobo et al., 1999; Nakamura et al., 2001; Lara et al.,
2003), that bind to either the G-box element (CACGTG) or ABA
response elements (ABREs) (Choi et al., 2000). Consistent with
its binding to the RY motif and its interaction with bZIP factors,
promoters of many ABI3-regulated genes contain RY motifs
coupled with a G-box or ABREs. Upon binding to these pro-
moters either directly or indirectly, ABI3 regulates the expression
of genes involved in seed dormancy, seed germination, desic-
cation tolerance, the accumulation of seed storage proteins, and
the breakdown of chlorophyll molecules in developing seeds.
The genetic networks operating under ABI3 to regulate these
processes are not fully understood.
We previously showed that SOMNUS (SOM) is a key direct
target of PIL5 that inhibits seed germination downstream of PIL5
(Kim et al., 2008).SOM encodes aC3H-type zinc finger protein of
unknown function. The expression analysis suggests that SOM
inhibits seed germination partly by activating the expression of
ABA synthetic genes and by inhibiting the expression of GA
synthetic genes downstream of PIL5 but not by activating the
expression of GAI and RGA in imbibed seeds. In this study, we
further investigated how the expression of SOM is regulated by
ABI3 and PIL5. We found that ABI3 directly binds to RY motifs
present in the SOM promoter and activates the expression of
SOM independently of PIL5 inmaturing seeds but collaboratively
with PIL5 in imbibed seeds. The interaction between ABI3 and
PIL5 at the protein level further indicates that these two key
regulators form a complex that regulates the expression of SOM
in imbibed seeds.
RESULTS
ABI3 Regulates the Expression of SOM
SOM inhibits seed germination by regulating genes downstream
of PIL5 that are involved in both GA and ABA metabolism in
imbibed seeds (Oh et al., 2007). We further investigated how the
expression of SOM is regulated. The public microarray data
compiled using theBARHeatMapper tool suggested thatSOM is
a seed-specific gene whose expression increases during seed
maturation (Figure 1A). The timing of SOM expression is slightly
later than that of ABI3 or FUS3 but similar to that of ABI5 or Em1
during seedmaturation. UnlikeABI3 andABI5,PIL5 is expressed
at a low level throughout seed maturation (Figure 1A).
Experimental analysis further supports that SOM is a seed-
specific gene. We determined the expression levels of SOM in
various tissues. SOM was expressed at very low levels in leaf,
root, stem, and flower tissues but at high levels in seeds. During
seed maturation, SOM began to be detected in siliques at 9 d
postanthesis (DPA), and its expression increased beyond this
ABI3 and PIL5 Collaborate to Regulate SOM 1405
time point (Figure 1B). The expression pattern of SOM during
seed maturation was similar to that of ABI5 and Em1, two seed-
specific genes that are known to be regulated by ABI3 (Figure
1C). When siliques were separated into developing seeds and
other parts (valve and replum), SOM was detected only in the
developing seeds but not in the valve and replum (Figure 1B,
inset), further supporting the seed-specific expression of SOM.
We analyzed the upstream region of SOM and noticed that
there were three RYmotifs, in addition to other motifs, such as an
ABRE and E-boxes, in the 2.7-kb intergenic region ranging from
the transcription start site of SOM to its upstream neighboring
gene (At1g03780) (Figure 1D). The three RY motifs are 0.3, 1.4,
and 2.4 kb away from the transcription start site of SOM. Since
the RY motif is bound by ABI3 or its homologs, the presence of
RYmotifs in its promoter suggests thatSOM is regulated by ABI3
or related B3-domain proteins during seed maturation.
To experimentally test whether ABI3 regulates the expression
of SOM during seed maturation, we determined the SOM tran-
script levels in an abi3 mutant. Since PIL5 regulates the expres-
sion of SOM in imbibed seeds, we first tested if PIL5 also
regulates the expression of SOM during seed maturation. As in
the wild type, however, the expression level of SOM increased
during seed maturation and reached a similar level in the pil5
mutant (Figure 2A), indicating that PIL5 does not play a signif-
icant role in regulating the expression of SOM during seed
maturation. In contrast with the pil5mutant, the expression level
ofSOM remained low in the abi3mutants during seedmaturation
and also in dry seeds (Figure 2A). These results indicate that ABI3
regulates the expression of SOM during seed maturation.
Seedmaturation is known to be regulated by three B3-domain
proteins (i.e., ABI3, FUS3, and LEC2) and a HAP3 subunit of
CCAAT binding protein (LEC1) (Giraudat et al., 1992; Luerssen
Figure 1. SOM Is Highly Expressed during Seed Maturation.
(A) Public micorarray data showing the seed-specific expression of SOM. The expression levels are visualized by BAR HeatMapper. Numbers beneath
the heat map indicate the relative expression levels, and the higher expression levels are indicated by more reddish colors. Ros., rosette; Caul., cauline;
Aging, aging rosette. Hours/Wc, imbibition hours in continuous white light.
(B) Seed-specific expression of SOM during seed maturation. The SOM mRNA expression levels were quantified by quantitative RT-PCR and are
indicated as relative expression levels compared with PP2A mRNA (SOM/PP2A) (SD, n = 3). An inset indicates the relative expression levels of SOM
mRNA in seeds and other tissues (valve and replum) that are separated from the 12-DPA silique.
(C) Relative expression of Em1 and ABI5 mRNAs during seed maturation (SD, n = 3).
(D) A diagram of the SOM promoter showing the RY motifs. G-box (CACGTG), PIL5-associated E-box (CANNTG), and ABRE (CACGTA) are also
indicated.
1406 The Plant Cell
et al., 1998; Raz et al., 2001; Stone et al., 2001). To investigate if
these other proteins also regulate the expression of SOM, we
determined the expression levels of SOM transcript in abi3, fus3,
lec1, and lec2 mutant seeds. The abi3 mutant seeds expressed
the lowest level of SOMmRNA. The fus3 and lec1 mutant seeds
also expressed lower levels of SOM than the wild type, while the
lec2 mutant expressed a similar level of SOM as the wild type
(Figure 2B). Since ABI3 levels were lower in the fus3 and lec1
mutants than in the wild type (Figure 2B, inset), the reduced
expression of SOM in the fus3 and lec1mutants could be due to
the lower levels of ABI3 in these mutants.
To further show that ABI3 is the major regulator of SOM, we
generated transgenic lines expressing eitherABI3 or FUS3 under
the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic
lines expressing FUS3 exhibited inflorescences with flowers that
opened prematurely and short and thick siliques. For the anal-
ysis, we selected homozygous transgenic lines that expressed
similar levels of ABI3 and FUS3 (ABI3-OX and FUS3-OX) (see
Supplemental Figure 1 online). We determined the expression
levels of the SOM transcript in transgenic leaves to avoid the
complex interlocked transcriptional regulation among ABI3 and
other seed maturation genes in the seeds (To et al., 2006). SOM
mRNA was expressed at higher levels in ABI3-OX but only
slightly higher levels in FUS3-OX (Figure 2C, inset). When the
plants were subjected to ABA treatment, the SOM expression
level increased dramatically only in the ABI3-OX line (Figure 2C),
supporting the notion that ABI3 activates the expression ofSOM.
ABI3 also activates the expression of luciferase in a transient
expression assay usingArabidopsis protoplasts and a 2-kbSOM
promoter linked to the luciferase gene (Figure 2D). Taken to-
gether, these results suggest that the seed-specific expression
of SOM is caused by the seed-specific expression of ABI3.
ABI3 is also required for the expression of SOM mRNA in
imbibed seeds. When imbibed, the level of SOM transcript
decreased drastically in the beginning of seed imbibition (after
3 h of seed imbibition) regardless of the light conditions. The
initial decrease in SOM transcript is in agreement with the initial
decrease in the majority of seed-specific mRNAs during seed
imbibition. After imbibition, however, the expression of SOM
depended on the light conditions. If a red light pulse was
administered at the beginning of seed imbibition (phyBon), the
SOM transcript level remained low, whereas it increased if a
far-red pulse was given (phyBoff) (Figures 3A and 3B). This light-
dependent expression of SOM in imbibed seeds, as reported
Figure 2. ABI3 Regulates the Expression of SOM.
(A) Decreased expression of SOM mRNA (SOM/PP2A) in the abi3-6 mutant but not in the pil5 mutant seeds.
(B) Relative expression of SOM mRNA (SOM/PP2A) in freshly harvested abi3-6 and other related mutant seeds. An inset indicates the relative
expression levels of ABI3 mRNA (ABI3/PP2A) in abi3-6 and its related mutant seeds.
(C) Increased expression of SOMmRNA (SOM/PP2A) in leaves of ABI3-OX. An inset indicates a magnified view of the relative expression levels of SOM
mRNA in the absence of ABA treatment.
(D) Transient expression assay showing the activation of the firefly luciferase reporter gene linked to the SOM promoter by ABI3 but not by FUS3 or GFP
in Arabidopis protoplasts. Error bars are SD (n = 3).
ABI3 and PIL5 Collaborate to Regulate SOM 1407
before (Kim et al., 2008), was regulated by PIL5, as the SOM
transcript levels remained low in the pil5 mutant, regardless of
the light conditions (Figure 3C). The SOM transcript levels
remained even lower in the abi3-sk11 mutant, regardless of the
light conditions (Figure 3D). These results indicate that ABI3 is
necessary for the high expression of SOMmRNA also in imbibed
seeds.
ABI3 Regulates the Expression of SOM by Directly Binding
to RYMotifs in the SOM Promoter in Vivo
The presence of RY motifs in the SOM promoter suggested that
ABI3 regulates the expression of SOM by directly binding to its
promoter. To determine if ABI3 directly binds to the SOM
promoter in vivo, we performed chromatin immunoprecipitation
(ChIP) analysis using imbibed seeds of a transgenic line ex-
pressing flag-tagged ABI3 under the control of the CaMV 35S
promoter. The flag-tagged ABI3 was functional, as it rescued the
green desiccation-intolerant seed phenotype of the abi3-6 mu-
tant. After the ChIP with anti-flag antibody, the enrichments of
specific SOM promoter fragments in the immunoprecipitates
were determined by six primer pairs (F1 to F6; Figure 4A). A
primer pair that amplifies the genomic region around the 5S rRNA
gene was included as a negative control, and two primer pairs
that amplify two seed-specific gene promoters containing RY
motifs (HsfA9 and 2S3) were also included as potential ABI3
target sites (Kagaya et al., 2005; Kotak et al., 2007).
The ChIP-enriched SOM promoter fragments ranging from F3
to F5 fragments, with the strongest enrichment being at the F5
fragment (Figure 4A). The HsfA9 and 2S3 promoters were also
enriched to a greater extent than the negative control, indicating
that ABI3 binds directly to the promoters of SOM, HsfA, and 2S3
in vivo. We noticed that the region of the SOM promoter that was
enriched by ABI3 is relatively wide (>1 kb), as if ABI3 binds to a
RYmotif (pRY) at 0.3 kb upstream of the transcription start site in
addition to a RYmotif (dRY) at 1.4 kb upstream. However, not all
fragments containing an RYmotif were enriched. A fragment (F1)
near an RYmotif at 2.4 kb upstream of the transcription start site
was not enriched much by ABI3, indicating that ABI3 binds to
only some RY motifs.
To determine if ABI3 can directly bind to an RYmotif present in
the SOM promoter, we performed electrophoretic mobility shift
assay (EMSA) analysis using a proximal RY motif–containing
double-stranded oligomer from the F5 fragment region. Recom-
binant ABI3 fused to maltose binding protein (MBP-ABI3) bound
to the oligomer, and its binding was effectively out-competed by
nonlabeled oligomer (Figure 4B). Unlike unlabeled oligomer,
however, a double-stranded oligomer containing a mutated RY
motif (mpRY) did not compete for the binding to MBP-ABI3.
Taken together, these results indicate that ABI3 binds to the
Figure 3. ABI3 Regulates the Light-Dependent Expression Levels of SOM mRNA in Imbibed Seeds.
(A) A diagram showing the light treatment schemes. Seeds were treated with either a far-red pulse (FRp, 2.8 mmol·m�2·s�1 for 5 min) or a far-red pulse
followed by a red pulse (Rp, 18.7 mmol·m�2·s�1 for 5 min) 1 h after the start of seed sterilization, before transferring the seeds to the dark.
(B) Light-dependent expression of SOM mRNA (SOM/PP2A) during imbibition of wild-type seeds (SD, n = 3).
(C) Abolition of light-dependent expression of SOM mRNA (SOM/PP2A) during imbibition of pil5 mutant seeds (SD, n = 3).
(D) Decreased expression of SOM mRNA (SOM/PP2A) during imbibition of abi3-sk11 mutant seeds (SD, n = 3).
1408 The Plant Cell
SOM promoter through RY motifs and activates the expression
of SOM.
To investigate the role of these RY motifs further in vivo, we
cloned a 2-kb region of the SOM promoter, fused it to a reporter
gene (b-glucuronidase [GUS]), and generated transgenic lines
(SOMpro:GUS). The 2-kb SOM promoter expressed the GUS
reporter gene in seeds and mildly in hypocotyls, vegetative
leaves, cauline leaves, and flowers (Figure 5A). When developing
embryos were dissected out, we observed that GUS expression
began to be detected in embryos 9 DPA and was strong in dry
seeds, consistent with the expression pattern of endogenous
SOM. Unlike the SOM promoter, the CaMV 35S promoter drove
expression of the GUS reporter gene throughout seedlings
(Figure 5A). The expression of SOM-green fluorescent protein
(GFP) under the same 2-kb SOM promoter also increased
dramatically in developing seeds. In imbibed seeds, the SOM-
GFP protein level decreased initially but increased again when
phyB was in an inactive state, consistent with the expression
pattern of endogenous SOM transcript in this condition (Figure
5B). These results indicate that the 2-kb region of the SOM
promoter linked to a reporter gene functionally substitutes the
endogenous SOM promoter.
To determine the role of RYmotifs in regulating the expression
of SOM, the 2-kb SOM promoter bearing mutations in two RY
motifs, either individually or together, was fused to a GUS
reporter gene and the respective transgenic lines were gener-
ated (mpRY:GUS, mdRY:GUS, and mpdRY:GUS). For each
promoter construct, 10 randomly chosen independent trans-
genic lineswere established, and the activities of promoterswere
determined by measuring the GUS transcript levels in the im-
bibed seeds. As expected, different transgenic lines expressed
different levels of GUS transcript; thus, we determined the
average expression levels of 10 transgenic lines. The average
expression level of GUS mRNA driven by wild-type 2-kb SOM
promoter was 38 relative to that of PP2A (Figure 5C). When a
distal or a proximal RY motif was mutated, the average relative
expression levels decreased to 10,whereaswhenbothRYmotifs
were mutated, the average relative expression level decreased
further to 5, indicating that two RY motifs contribute similarly to
the high expression of SOM. Consistent with the view that both
RY motifs contribute to SOM expression, the relative expression
level of SOMwas reduced when a T-DNA fragment was inserted
between the proximal and distal RY motifs (som-4 allele),
whereas the level was further reduced when a T-DNA fragment
was inserted between the proximal RY motif and the transcrip-
tion start site (som-3 allele) (Figure 5D). Taken together, these
results indicate that both RY motifs are required for the high
expression of SOM.
ABI3 Interacts with PIL5 to Potentiate the Expression of
SOM in Imbibed Seeds
Our analysis indicates that the expression of SOM mRNA is
regulated by both ABI3 and PIL5 in imbibed seeds. In imbibed
seeds, public microarray data show that PIL5 is expressed
comparably in both tissues, while ABI3 is expressed higher in
endosperm. Consistent with the requirement of both ABI3 and
PIL5 for the expression of SOM, SOM is also expressed higher in
endosperms than in embryos of imbibed seeds (see Supple-
mental Figure 2 online). We further investigated the role of ABI3
and PIL5 in regulating the expression of SOM by comparing the
expression levels of SOM in variousmutants in imbibed seeds. In
the wild type, the expression levels of SOM transcript were;5-
fold higher under the phyBoff condition than the phyBon condition
(Figure 6A). In the pil5 mutant, the expression levels of SOM
transcript were similarly low, regardless of the light conditions,
consistent with a previous report that showed that PIL5mediates
light signaling to regulate the expression of SOM mRNA (Kim
Figure 4. ABI3 Directly Binds to the SOM Promoter through RY Motifs
Both in Vivo and in Vitro.
(A) ChIP analysis showing the direct binding of ABI3 to the SOM
promoter in vivo. Top diagram, the SOM promoter and three RY motifs
(a proximal RY motif [pRY], a distal RY motif [dRY], and an RY motif near
At1g03780 [RY]). Seeds imbibed for 12 h under phyBoff conditions were
used for ChIP analysis. F1 to F6 indicate genomic DNA fragments around
the SOM promoter tested for enrichment by quantitative PCR (SD, n = 3).
IP indicates immunoprecipitated DNA, and IN indicates input DNA.
(B) EMSA analysis showing the direct binding of ABI3 to pRY in the SOM
promoter in vitro. MBP-ABI3, ABI3 protein fused to MBP; cold probe,
nonlabeled wild-type pRY (pRY) or mutated pRY (mpRY). The increased
concentration of competitors (53, 103, and 203) is indicated by
triangles.
ABI3 and PIL5 Collaborate to Regulate SOM 1409
et al., 2008). In the abi3 mutants, the expression levels of SOM
mRNA were greatly reduced relative to the wild type, indicating
that ABI3 is also necessary for the high expression of SOM
(Figure 6A). However, the SOMmRNA was still expressed 6-fold
higher in FR-treated seeds than in R-treated imbibed seeds of
the abi3mutant, indicating that ABI3, although necessary for the
high expression of SOM, is dispensable for the light-dependent
expression of SOM mRNA. This reduced expression of SOM in
the abi3mutant was not due to the lower levels of PIL5 transcript
(Figure 6B). Taken together, these results suggest that PIL5
regulates the light-dependent expression of SOMmRNA even in
the absence of ABI3, while ABI3 collaborates with PIL5 to in-
crease the expression of SOM mRNA in imbibed seeds.
Numerous transcription factors have been shown to regulate
the expression of target genes collaboratively by forming a
complex on the promoters of the target genes. For example,
ABI3 has been shown to interact with a subset of bZIP tran-
scription factors, including ABI5, bZIP10, and bZIP25, in Arabi-
dopsis (Nakamura et al., 2001; Lara et al., 2003). In maize, VP1,
an ABI3 homolog, also interactswith TRAB1, a bZIP transcription
factor (Hobo et al., 1999). We investigated if ABI3 and PIL5
interact with each other at the protein level. We detected the
interaction by an in vitro binding assay using MBP-tagged ABI3
and His-tagged PIL5. As a positive control, we included His-
tagged ABI5, a known ABI3-interacting transcription factor.
WhenMBP-ABI3 was precipitated, it coprecipitated PIL5 (Figure
7A), whereas MBP alone did not coprecipitate PIL5, indicating
that ABI3 interacts with PIL5. Consistent with a previous report,
MBP-ABI3 but not MBP alone coprecipitated ABI5. The results
indicate that ABI3 interacts with various transcription factors,
including PIL5 and ABI5, at the protein level.
The physical interaction between ABI3 and PIL5 may suggest
that they help each other to bind to the SOM promoter. Alterna-
tively, they may bind to the SOM promoter independently but
interact with each other to activate the expression of SOM
mRNA. The light-dependent expression of SOM mRNA in the
Figure 5. RY Motifs Are Necessary for the High Expression of SOM.
(A) Seed-specific expression driven by a 2-kb region of the SOM promoter. SOMpro:GUS indicates various tissues and organs of transgenic plants
harboring SOMpro:GUS, while 35Spro:GUS indicates transgenic plants harboring 35Spro:GUS. Numbers and DS on figures indicate DPA and dry seeds,
respectively. Bar = 5 mm.
(B) Immunoblot analysis showing the increased expression of SOM-GFP protein by the SOM promoter during seed maturation and imbibition. hours,
hours after imbibition.
(C) Decreased expression of GUS reporter transcripts (GUS/PP2A) by the SOM promoter with mutations in their RY motifs. Numbers indicate
average 6 SD (n = 10). Seeds imbibed for 24 h under phyBoff condition were used for analysis. The inset shows the light-dependent expression of the
GUS transcript in two transgenic lines. Error bars are SD (n = 3).
(D)Decreased expression of SOMmRNA (SOM/PP2A) in the som-4 allele. Seeds imbibed for 12 h were used for analysis. Triangles indicate RYmotifs in
the SOM promoter (SD, n = 3).
1410 The Plant Cell
abi3 mutant suggested that PIL5 binds to the SOM promoter
even in the absence of ABI3 (Figure 6A). We further determined if
ABI3 and PIL5 affect the binding of each other to the SOM
promoter in vivo. ChIP analysis was used to determine the
binding of ABI3 and PIL5 to the SOM promoter in vivo in the
presence or absence of PIL5 or ABI3. The ChIP analysis showed
that ABI3 binds to the SOM promoter similarly both in ABI3-OX
and ABI3-OX/pil5 (Figure 7B), while PIL5 also binds to the SOM
promoter similarly both inPIL5-OX andPIL5-OX/abi3 (Figure 7C),
indicating that ABI3 and PIL5 bind to the SOM promoter even in
the absence of each other. Taken together, these results indicate
that PIL5 and ABI3 do not drastically affect each other’s ability to
bind to the SOM promoter in vivo. Rather, they suggest that they
interact with each other to activate the expression of SOM.
DISCUSSION
We show that ABI3 binds to the SOM promoter through two RY
motifs usingChIP analysis andEMSA. Binding of ABI3 to bothRY
motifs was necessary to activate the expression of SOM mRNA
fully, as mutations in either of two RY motifs greatly reduced the
expression of a reporter gene. We analyzed the relationship
between ABI3 and PIL5, which also regulates the expression of
SOM by directly binding to its promoter (Kim et al., 2008). We
found that ABI3 and PIL5 regulate the expression of SOM either
independently or collaboratively. In maturing seeds, ABI3, but
not PIL5, was necessary for the high expression of SOM mRNA,
whereas, in imbibed seeds, PIL5 mediated light-dependent
expression of SOM mRNA and ABI3 potentiated the ability of
PIL5 to increase the expression level of SOM mRNA. At the
protein level, ABI3 interacts with PIL5. This protein–protein
interaction, however, did not drastically affect the targeting of
ABI3 to theSOM promoter, indicating that ABI3 and PIL5 interact
with each other to regulate SOM transcription collaboratively in
imbibed seeds. Since ABI3 and PIL5 are key signaling compo-
nents of ABA signaling and light signaling (Lopez-Molina et al.,
2002; Oh et al., 2007), respectively, our results suggest that the
SOM promoter integrates ABA and light signaling to regulate
seed germination.
Figure 6. PIL5 Requires ABI3 to Activate the Expression of SOM
Efficiently.
(A) The expression levels of SOM mRNA (SOM/PP2A) in the wild type,
the pil5 and abi3-sk11 mutants, and the abi3-sk11 pil5 double mutant.
Seeds were imbibed for 12 h. The inset indicates the fold difference in
SOM mRNA expression between the phyBoff and phyBon conditions (SD,
n = 3). Student’s t test: **P < 0.01.
(B) Expression levels of PIL5mRNA (PIL5/PP2A) in the wild type, the pil5
and abi3-sk11 mutants, and the pil5 abi3-sk11 double mutant (SD, n = 3).
Figure 7. ABI3 and PIL5 Interact at the Protein Level.
(A) In vitro binding assay showing the protein–protein interaction be-
tween ABI3 and PIL5. MBP-ABI3, MBP-fused ABI3 protein; PIL5H, His-
tagged PIL5 protein; ABI5H, His-tagged ABI5 protein; IN, 5% of input
proteins.
(B) ChIP assay showing the similar enrichments of SOM promoter
fragments by ABI3-flag both in the ABI3-OX and ABI3-OX pil5 lines. F1
and F5 are as indicated in Figure 4A. The values shown are immuno-
precipitated DNA/input DNA relative to F1. Error bars are SD (n = 3).
(C) ChIP assay showing that PIL5-myc enriches SOM promoter frag-
ments to a similar extent both in the PIL5-OX3 and PIL5-OX3 abi3-6 lines.
F1 and F5 are as indicated in Figure 4A. The values shown are
immunoprecipitated DNA/input DNA relative to F1 (SD, n = 3).
ABI3 and PIL5 Collaborate to Regulate SOM 1411
ABI3 Is Necessary for the High Expression of SOM Both in
Maturing and Imbibed Seeds, but PIL5 Is Necessary Only
in Imbibed Seeds
Our expression analysis shows that ABI3 is necessary for the
activation of SOMmRNA both in maturing seeds and in imbibed
seeds. However, PIL5 was necessary only in the imbibed seeds,
but not in the maturing seeds. What determines this stage-
specific role of PIL5 is not clear. A few different possible expla-
nations may account for the stage specificity. First, maturing
seeds may express other PIF family members that activate the
expression of SOM mRNA redundantly with PIL5. Public micro-
array data indicate that PIL5 is expressed at lower levels than
other PIFs (PIF5 and PIF6) in maturing seeds, while PIL5 is a
dominant PIF in imbibed seeds (see Supplemental Figure 3
online). If other PIFs could activate the expression of SOM
redundantly with PIL5, the mutation in PIL5 would be phenotyp-
ically manifested more severely in imbibed seeds than in matur-
ing seeds. Second, other transcription factors that are known to
interact with ABI3may functionally substitute for PIL5 inmaturing
seeds. The same public microarray data indicate that ABI5, EEL,
DPBF2, and AREB3 are expressed at high levels in maturing
seeds (see Supplemental Figure 3 online) (Bensmihen et al.,
2005). Since these bZIP transcription factors are known to
activate the expression of seed storage-specific protein genes
in maturing seeds, it is possible that these bZIP factors bind to
the SOM promoter and activate the expression of SOM in
maturing seeds. Alternatively, stage-specific modification or
cofactors that enable ABI3 to activate the expression of SOM
mRNA alone may account for the dispensability of PIL5 in
maturing seeds. Further analysis is needed to clarify why PIL5
is dispensable for the activation of SOM mRNA in maturing
seeds.
ABI3 Binds to the SOM Promoter through RYMotifs to
Activate the Expression of SOMmRNA
A previous study showed that SOM is a C3H-type zinc finger
protein that inhibits seed germination downstream of PIL5 partly
by activating ABA biosynthesis and inhibiting GA biosynthesis in
imbibed seeds (Oh et al., 2007; Kim et al., 2008). We further
investigated how the expression of SOM is regulated in seeds.
Public microarray data indicated that SOM is highly expressed in
maturing seeds, which is reminiscent of the expression pattern of
ABI3-regulated genes, such asABI5 (Figure 1A). The presence of
RY motifs, which serve as a binding site of B3-domain proteins,
such as ABI3, FUS3, and LEC2, further suggested that SOM is
regulated by ABI3 or its homologs. Consistent with this notion,
the expression level of SOM was drastically reduced in abi3 and
weakly reduced in fus3. Transgenic lines overexpressing ABI3
and FUS3 showed that the expression of SOM mRNA is acti-
vated mainly by ABI3. Apparently, ABA strongly enhanced the
expression of SOM by ABI3. Taken together, our results suggest
that the high expression of SOM in maturing seeds is caused by
the seed-specific expression of ABI3 and the concomitant in-
crease in ABA level during seed maturation.
Our ChIP analysis coupled with EMSA indicated that ABI3
directly binds to two regions of the SOM promoter through RY
motifs and activates the expression of SOMmRNA. The binding
of ABI3 to both RY motifs was necessary for the high expression
ofSOMmRNA, asmutations in either one of theRYmotifs greatly
decreased the expression levels of a reporter gene. As in the
SOM promoters, multiple RY motifs are also found in the pro-
moters ofmany seed storage protein genes, includingOLEOSIN,
CRUCIFERIN, and 2S, suggesting that promoters of ABI3-
regulated genes recruit multiple copies of ABI3. Interestingly,
an insertion of a 4.5-kb T-DNA fragment from pBIN-pROK2 be-
tween two of the RY motifs also reduced the expression of SOM
mRNA. Since the insertion of the T-DNA fragment increases the
distance between the distal RY motif and the transcription start
site, the reduced expression may indicate that ABI3 must be in
relatively close proximity to the transcription start site to activate
the transcription. Alternatively, the insertion of T-DNA may dis-
rupt the molecular interaction among proteins that bind to the
proximal region and proteins that bind to distal regions of the
SOM promoter.
The ChIP analysis further indicates that ABI3 binds to only a
subset of RY motifs in vivo. A genomic region we scanned by
ChIP analysis included three RY motifs. Among these RY motifs,
only two were highly enriched, and one was not enriched by
ABI3. The enrichment was not correlated with the distance from
the transcription start site, as two enriched RY motifs were 0.3
and 1.4 kb upstream of the SOM transcription start site, respec-
tively, while one nonenriched RY motif was 0.3 kb upstream of
the transcription start site of At1g03780 (Figure 1D). These
results indicate that ABI3 binds to only a subset of RY motifs in
vivo and imply that in vivo ABI3 binding sites will have to be
determined experimentally, such as by ChIP analysis. Binding of
the transcription factor to its target appeared to be more selec-
tive under in vivo conditions than in vitro, and this seems to be a
rather general phenomenon, instead of an exception. A recent
ChIP-Chip analysis showed that PIL5 binds to only a subset of
G-box elements in vivo (Oh et al., 2009). Similarly, a bZIP
transcription factor, HY5, binds to only a subset of G-box
elements (Lee et al., 2007). The molecular mechanisms that
determine how the in vivo binding sites are selected for each
transcription factor are not known.
ABI3 and PIL5 Interact and Activate the Expression of SOM
mRNA Collaboratively in Imbibed Seeds
ABI3 and PIL5 collaboratively activated the expression of SOM
mRNA. The expression ofSOMmRNAwas greatly reduced in the
abi3 mutant. Mutations in RY motifs greatly decreased the
expression of reporter genes, supporting the hypothesis that
ABI3 activates the expression of SOM. Nevertheless, the ex-
pression of SOMmRNA was still repressible by red light, as long
as PIL5 was present, indicating that PIL5mediates light signaling
to activate the expression of SOM mRNA, while ABI3 enhances
the expression level.
Although the molecular mechanisms underlying the ability of
ABI3 to enhance the activity of PIL5 are not known, the interac-
tion between ABI3 and PIL5 at the protein level may contribute to
this collaboration. ABI3 and PIL5 may form a protein complex
that activates the transcription more efficiently. Alternatively,
ABI3 and PIL5 may help each other bind to the SOM promoter.
1412 The Plant Cell
Our data indicate that ABI3 and PIL5 do not drastically affect
each other’s targeting to the SOM promoter, implying that the
synergism comes after their targeting to the promoter, rather
than during the targeting itself (Figures 6 and 7). Our ChIP
analysis indicates that ABI3 enriches the SOM promoter and that
this enrichment is of a similar magnitude in the wild type and the
pil5 mutant. PIL5 also enriched the SOM promoter, and this
enrichment was similar in the wild type and the abi3 mutants.
These results indicate that ABI3 and PIL5 can bind to their target
sites in the absence of each other. Our results are similar to a
previous report showing that ABRE binding factors bind to the
maize rab28 promoter even in the absence of VP1 (Busk and
Pages, 1997), while they are different to another report showing
that ABI3 slightly enhances the binding between bZIP10/OPA-
QUE2 and the G-box element in vitro (Lara et al., 2003). Since a
slight increase or decrease in binding affinity is difficult to resolve
in our ChIP assay, the results do not exclude the possibility that
ABI3 mildly affects the ability of PIL5 or other interacting bZIP
factors to bind to DNA. Therefore, even though ABI3 and PIL5 do
not drastically affect each other’s binding to SOM promoter,
further analysis is needed to determine if they affect DNA binding
activity more subtly.
The direct interaction between ABI3 and PIL5 indicates that
three germination-inhibiting transcription factors (PIL5, ABI3,
and ABI5) form a functional module, both through transcriptional
regulation and protein–protein interaction, to inhibit seed germi-
nation. At the transcriptional level, PIL5 directly binds to pro-
moters of ABI3 and ABI5 and activates the expression of their
mRNAs, while ABI3 activates the expression of ABI5 mRNA. At
the protein level, ABI3 interacts with both its regulator (PIL5) and
its regulatee (ABI5), which leads to the synergistic activation of
their target gene expression. This type of functional module that
is composed of interconnected transcription factors is likely to be
useful to regulate the expression of wide sets of genes either
independently, in combination with each other or with other
proteins as well, or interdependently. Further studies are needed
to decipher which target genes are regulated by these three
factors independently, in combination, or interdependently.
METHODS
Plant Materials and Growth Conditions
Arabidopsis thaliana plants were grown in a growth room with a 16-h-
light/8-h-dark cycle at 22 to 248C. T-DNA insertion lines (abi3-sk11, som-
4, lec1, and lec2) were obtained from the Salk Institute (Salk_023411,
CS806302, Salk_131219, and CS873714, respectively), and insertion
sites of abi3-sk11 and som-4 were determined by amplifying and se-
quencing the flanking regions. The lec1 and lec2mutants were confirmed
by monitoring anthocyanin accumulation and desiccation intolerance in
seeds. To generate ABI3-OX and FUS3-OX plants, full-length ABI3 and
FUS3 cDNAswere amplified, cloned into binary vectors, and transformed
into Arabidopsis, and then homozyogous lines were selected (primers are
presented in Supplemental Table 1 online). For transgenic lines harboring
the GFP-tagged mini-gene construct of SOM (SOMpro:SOM-GFP) in the
som-2background,SOM cDNA and its promoter regionwere sequentially
cloned into the pbGFP1 binary vector and transformed into the som-2
mutant. Homozygous lines were selected. For mutagenesis of the RY
motifs in the SOM promoter, inverse PCR was performed with mutated
primer sets (see Supplemental Table 1 online) using a SOM promoter-
containing plasmid as a template. Mutated SOM promoters were
confirmed by sequencing, cloned into a pbGG4 binary vector for pro-
moter-reporter construction, and transformed into Columbia-0 (Col-0).
Ten independent transgenic lines showing the 3:1 segregation ratio were
used to monitor the expression of GUS mRNA during imbibition. The
abi3-6 and fus3-3 mutant seeds were kind gifts from F. Parcy at Centre
National de la Recherche Scientifique, France. All themutants used in this
study (som-2, som-3, som-4, pil5-1, PIL5-OX3, lec1, lec2, fus3-3, abi3-
sk11, and abi3-6) are in the Col-0 background. Desiccation-intolerant
mutants weremaintained by sowing seeds harvested from green siliques.
Quantitative and Qualitative Gene Expression Analysis
For mRNA expression analysis, total RNAs were extracted either from
dried seeds, imbibed seeds, or transgenic leaves using a Spectrum plant
total RNA kit (Sigma-Aldrich) and converted to cDNAs using MMLV-
RTase (Promega) according to the manufacturers’ protocols. Transcript
levels of each mRNA were determined by real-time PCR and normalized
with the level of PP2A mRNA using the delta Ct method (Czechowski
et al., 2005; Schmittgen and Livak, 2008). Amplification log curves of each
primer set were confirmed to be parallel to that of PP2A amplification to
validate the comparisons among samples and primer sets before use.
Real-time PCR was performed with a protocol (45 cycles, each cycle
consisting of 958C/588C/728C for 20 s each) in an iCycler iQ5 cycler (Bio-
Rad). We tested more than three different seed batches to confirm the
gene expression patterns and presented representative results in each
figure.
Expression browser andHeat-Mapper tools provided by BAR (The Bio-
Array Resource for Arabidopsis Functional Genomics; http://bbc.botany.
utoronto.ca/) were employed to show the heat map of gene expression
patterns.
To determine the tissue-specific expression of SOM by histochemical
GUS reporter assay, seed coats, which inhibit penetration of X-Gluc into
seeds, were carefully pinched off the mature or imbibed seeds using
forceps. Decoated seed embryos were stained for GUS activity after
having undergone fixing in 90% acetone for 30 min, as described
previously (Jefferson et al., 1987).
For the effector activity assay of ABI3 and FUS3 using Arabidopsis
protoplasts, mesophyll protoplasts were prepared from fully mature
leaves as described previously (Yoo et al., 2007). Briefly, leaf slices
were dipped in cell wall digestion solution (10 mM MES, pH 5.7, 1.5%
cellulase R10, 0.4% macerozyme R10, 0.4 M mannitol, 20 mM KCl, 10
mM CaCl2, and 0.1% [w/v] BSA) and incubated for 3 h with mild shaking.
One hundred microliters of protoplasts (2 3 104) was used for each
transfection. After incubation for 1 d, luciferase activity was measured
using a Dual Luciferase assay kit (Promega).
For the analysis of protein levels, samples were ground in protein
extraction buffer (100 mMNaH2PO4, 8 M urea, and 10mM TrisCl, pH 8.0)
using Tissue-lyzer (Qiagen). Tissue lysates were cleared by centrifugation
at 20,000g for 10 min and subjected to immunoblotting as described
previously (Park et al., 2004).
In Vitro Binding Assay
For the in vitro binding assay between PIL5 and ABI3, recombinant MBP-
ABI3 protein was produced in Escherichia coli at 258C using the pMAL-
c2X-ABI3 expression vector and purified using amylose-resin following
the manufacturer’s protocol (NEB) (see Supplemental Table 1 online for
primer sets). His-tagged PIL5 and ABI5 (PIL5H and ABI5H) were pro-
duced as described previously (Oh et al., 2007). MBP-resin and MBP-
ABI3-resin were incubated with purified PIL5H and ABI5H at 48C for 2 h,
sedimented by spin down, andwashed five times.MBP resin was used as
ABI3 and PIL5 Collaborate to Regulate SOM 1413
an MBP control as well as a nonspecific binding control. Then, 13 PBS
with 0.1% Nonidet P-40, 0.5 mM DTT, 10% glycerol, 1 mM PMSF, and
Protease inhibitor cocktail (Roche) was used for binding and washing.
PIL5H and ABI5H bound to MBP-ABI3 were visualized by protein blotting
using anti-His antibody.
ChIP Analysis and EMSA
ChIP was performed essentially as described previously, except that
three separate steps (the elution of the DNA–protein complex from resin,
the reversal of cross-linking, and the digestion with proteinase K) were
combined into one step in this study (Kim et al., 2008). Immunoprecipi-
tated DNA was eluted from resin in 200 mL of elution buffer containing 20
mM Tris-HCl, pH 7.5, 5 mM EDTA, 50 mM NaCl, 1% SDS, and 50 mg/mL
proteinase K at 688C for 2 h with rotary mixing. Residual DNA was further
eluted by subsequent incubation in 100 mL of elution buffer at 688C for 5
min. Eluted DNA was purified with a PCR purification kit (Solgent) and
subjected to real-time PCR. For ChIP of flag-tagged ABI3, anti-flag
antibody-conjugated resin (Sigma-Aldrich) was used, while anti-myc Ab
(Cell Signaling) and protein A–conjugated resin (Upstate) was used for
ChIP of myc-tagged PIL5. For EMSA, previously described procedures
were followed, except that 59 biotin-labeled probes were synthesized by
chemical modification (Bioneer).
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: PIL5 (At2g20180), ABI3 (At3g24650), SOM (At1g03790), LEC1
(At1g21970), LEC2 (At1g28300), FUS3 (At3g26790), ABI5 (At2g36270),
Em1 (At3g51810), Em6 (At2g40170), 2S3 (At4g27160), RGA (At2g01570),
HsfA9 (At5g54070), 5S rRNA (At3g41979), and PP2A (At1g13320).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Expression Levels of ABI3 and FUS3
Transgenes.
Supplemental Figure 2. Tissue-Specific Expression of ABI3, PIL5,
and SOM.
Supplemental Figure 3. Expression of PIFs and bZIPs during Seed
Maturation and Imbibition.
Supplemental Table 1. Primers Used in This Study.
ACKNOWLEDGMENTS
This work was supported in part by grants from the National Research
Foundation of Korea (2007-0056949 and 2009-0077873) to G.C.
Received October 25, 2010; revised February 12, 2011; accepted March
25, 2011; published April 5, 2011.
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ABI3 and PIL5 Collaborate to Regulate SOM 1415
DOI 10.1105/tpc.110.080721; originally published online April 5, 2011; 2011;23;1404-1415Plant Cell
Jeongmoo Park, Nayoung Lee, Woohyun Kim, Soohwan Lim and Giltsu Choi SeedsArabidopsisPromoter in Imbibed
by Directly Binding to ItsSOMNUSABI3 and PIL5 Collaboratively Activate the Expression of
This information is current as of April 16, 2021
Supplemental Data /content/suppl/2011/04/06/tpc.110.080721.DC1.html
References /content/23/4/1404.full.html#ref-list-1
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